Abstract Systems Genetics Analysis of Tumor Evolution in the Mouse. Metastasis is the major cause of death due to cancer in humans, but many factors play a role in determining the route taken by initiated cells to reach this end stage. We know that tumors arise due to the combined effects of inherited genetic factors and environmental exposures, and have taken the long term view that successful modeling of human cancer in the mouse requires us to take account of interactions between genetics and environment. The over-arching goal of this research is to develop an integrated systems genetics view of the cells, signaling pathways and mutations in specific genes involved in evolution of metastasis from single initiated cells, using one of the best characterized models of multistage carcinogenesis of the skin. Based on major funding from the NCI MMHCC over the past 15 years, we have built up a unique tissue and tumor bank comprising thousands of samples of Ras-mutant squamous tumors from a genetically heterogeneous mouse population. These samples encompass all stages from benign lesions to metastases, and in particular include hundreds of matched primary carcinomas and distant metastases from the same animals. All tumors were induced by exposure to mutagens and tumor promoting agents, resulting in complex genomic landscapes that resemble, in mutation frequency and type, the genomic profiles of human squamous carcinomas of the skin, head and neck, and lung. All mice in the cohort have been genotyped genome-wide to facilitate linkage and eQTL analysis to identify genetic determinants of initiation, benign tumor formation, progression and metastatic dissemination. This project will exploit this unique tissue bank and database by using genomic technologies, together with novel mouse strains, to identify the cells of origin of benign and malignant tumors, biomarkers of risk of malignant progression, and genetic drivers of metastasis. Computational network analysis tools will be used to study the evolution of signaling pathways, for example the Ras pathway, through multiple stages of carcinogenesis, to identify changes in these networks that will identify potential novel cancer target genes. Finally we will initiate a new series of translational studies of immunotherapy, based on our observation that these chemically induced tumors harbor novel neo-antigens and show promising responses to inhibitors of immune checkpoint proteins.